An important mechanism regulating protein synthesis in many organisms in phosphorylation of eukaryotic initiation factor-2 (eIF-2). Many different stress signals have been identified that elicit eIF-2 phosphorylation, including viral infection, amino acid or serum deprivation, iron deficiency, calcium mobilization and cerebral ischemia. In addition, inappropriate regulation of eIF-2 phosphorylation was found to lead to malignant transformation. The goals of this proposal are twofold. The first goal focuses on the regulation of an eIF-2 kinase from yeast, GCN2. GCN2 senses amino acid starvation in yeast and transduces this signal, via phosphorylation of eIF-2, to stimulate translation of GCN4, a transcriptional activator of genes involved in amino acid biosynthesis. Sequences in GCN2 homologous with histidyl-tRNA synthetases (HisRSs) stimulate kinase activity by interacting with uncharged tRNAs that accumulate during amino acid starvation. We will address the roles of ribosome targeting and autophosphorylation and the specificity of the HisRS-related domain in the activation of GCN2 kinase. The second goal of this proposal is to understand the molecular basis for reduced initiation of protein synthesis in mammalian cells starving for amino acids. Given the important role of GCN2 in translational control in lower eurkaryotes, we searched for and identified a mammalian cDNA with homology to yeast GCN2. This new mammalian kinase contains both kinase and HisRS-related domains and is expressed in a variety of tissues, including liver and brain, as judged by Northern blot analysis. We propose that phosphorylation of eIF-2 by GCN2 kinase reduces translation initiation in starving mammalian cells. To achieve the two stated goals, we will address four specific aims. 1) Characterize the ribosomal binding site required for yeast GCN2 kinase stimulation of translation control. 2) Characterize the roles of phosphorylation and the HisRS-related domain in the activation of yeast GCN2 kinase by uncharged tRNA levels. 3) Characterize the role of mammalian GCN2 in translation control using yeast as a model system. 4) Characterize the role of GCN2 kinase in the phosphorylation of eIF-2 in mammalian cells starving for amino acids. These studies will further our understanding to the mechanisms by which tRNA and ribosome interaction facilitate phosphorylation of eIF-2 by GCN2 kinase. Additionally, these studies will determine the role of the new mammalian GCN2 kinase in the control of protein synthesis in nutrient-deprived mammalian cells.